Size-Controllable Prussian Blue Nanoparticles Using Pluronic Series for Improved Antioxidant Activity and Anti-Inflammatory Efficacy

Abstract

Prussian blue (PB) is a metal cluster nanoparticle (NP) of cyanide-bridged iron(II)-iron(III) and exhibits a characteristic blue color. Its peroxidase-, catalase-, and superoxide-dismutase-like activities effectively remove excess reactive oxygen species that induce inflammation and tumorigenesis. However, the dispersion of PB NPs is not sufficiently stable for their application in the biomedical field. In this study, we developed Pluronic-stabilized Prussian blue nanoparticles (PB/Plu NPs) using a series of Pluronic triblock copolymers as a template material for PB NPs. Considering the hydrophilic-lipophilic balance (HLB) values of the Pluronic series, including F68, F127, L35, P123, and L81, the diameters of the PB/Plu NPs decreased from 294 to 112 nm with decreasing HLB values. The smallest PB NP stabilized with Pluronic P123 (PB/PP123 NP) showed the strongest antioxidant and anti-inflammatory activities and wound-healing efficacy because of its large surface area. These results indicated that the spatial distribution of PB NPs in the micelles of Pluronic greatly improved the stability and reactive oxygen species scavenging activity of these NPs. Therefore, PB/Plu NPs using U.S.-FDA-approved Pluronic polymers show potential as biocompatible materials for various biomedical applications, including the treatment of inflammatory diseases in the clinic.

Keywords: Pluronic; Prussian blue; anti-inflammation; antioxidant; reactive oxygen species; wound healing.

Figure 1

Schematic preparation of novel Prussian blue nanoparticles stabilized by Pluronic polymers (PB/Plu NPs). HLB, hydrophilic–lipophilic balance.

Figure 2

Characterization of Prussian blue nanoparticles (PB NPs) and Pluronic-stabilized Prussian blue nanoparticles (PB/Plu NPs). (a) Hydrodynamic diameters; (b) polydispersity index (PDI); (c) zeta potentials; (d) photographs of the PB NP and templates used (Pluronic series including F68, F127, L35, P123, and L81); (e) UV–Vis absorption spectra of PB NP and PB/Plu NPs. ▲ indicates partial aggregation of nanoparticles.

Figure 3

Physicochemical properties of PB NP and PB/Plu NPs. (a) Transmission electron microscopy images of (i) PB NP, (ii) PB/PF127 NP, and (iii) PB/PP123 NP (scale bar = 200 nm). Insets are highly magnified images with scale bars of 50 nm. (b) Fourier-transform infrared (FT-IR) spectra of PB NP, PB/PF127 NP, and PB/PP123 NP.

Figure 4

Stability analysis of PB NP and PB/Plu NPs in aqueous solution at 37 °C. (a) Hydrodynamic diameters; (b) polydispersity index (PDI); (c) zeta potential after freeze drying (FD) and 4 weeks of storage at 37 °C. ▲ denotes partial aggregation of the nanoparticles.

Figure 5

In situ antioxidant activity of PB NP and PB/Plu NPs. (a) 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and (b) hydroxyl radical scavenging assay of PB NP and PB/Plu NPs. *** p < 0.001.

Figure 6

In vitro cytotoxicity, antioxidant activity, and anti-inflammatory activity of PB/PP123 NP. (a) Cytotoxicity analysis of 0.01–5 mg/mL NPs, (b) antioxidant effect of NPs (control (CTL) indicates the lowest reactive oxygen species (ROS) level), and (c) anti-inflammatory efficacy (CTL indicates the lowest nitric oxide level). # p > 0.05 and * p < 0.05.

Figure 7

In vitro wound-healing efficacy of PB/PP123 NP. (a) Wound closure of NIH 3T3 fibroblast cells after treatment with NPs at different time points, and (b) wound-healing activity of NPs at different concentrations after 24 h of treatment (* p < 0.05, ** p < 0.01, and *** p < 0.001).